Microsatellites, or simple sequence repeats (SSRs) are regions of DNA that contain short tandem repeats (STRs) of 1 to 6 nucleotides. Microsatellites occur ubiquitously in all prokaryotic and eukaryotic genomes (Buschiazzo and Gemmell, 2006; Kelkar et al., 2008) and are popular markers for population genetics (Guichoux et al., 2011). Microsatellite markers are one of the most informative and versatile DNA-based markers used in genetic research, however, their development has traditionally been a costly process. Recent advances in next generation sequencing technologies allow the efficient identification of large numbers of microsatellites (Hudson, 2008; Morozova and Marra, 2008) at a relatively low cost and effort of traditional approaches. NGS method produce large amount of sequence data and are used to isolate and develop numerous genome wide and gene based microsatellite loci. We at MRDNA use Illumina MiSeq platform for microsatellite identification. Sequencing and microsatellite identification steps (figure1) includes isolation and purification of genomic DNA, fragmentation, ligation to sequencing adapters and purification following the standard protocol of the Illumina TruSeq DNA Library Kit. Following the denaturation and amplifications steps libraries can be pooled and sequenced. The resulting reads are analyzed with the program PAL_FINDER_v0.02.03 (Castoe et al., 2012) to extract those reads that contain perfect 2mer, 3mer, 4mer, 5mer, and 6mer tandem SSRs. Reads are identified as SSRs if they contain simple repeats of at least 12 bp in length for 2–4mers (e.g., 6 tandem repeats for dinucleotides), and at least 3 repeats for 5mers or 6mers. The reads are then sorted by the monomer sequence of the repeat (e.g., TAC or TA repeats) and by the number of tandemly repeated units. The program is operated using a control file that determines parameter settings. A number of recent studies demonstrate the efficient use of Illumina technologies for the discovery of microsatellites in various organisms (Zalapa et al., 2012; Nunziata et al., 2012; Castoe et al., 2012).We at MR DNA routinely perform DNA sequencing and microsatellite identification and provide cost effective high quality data and robust output from only little amount of input DNA.
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